Crystallization and preliminary X-ray crystallographic analysis of spruce budworm antifreeze protein.

Antifreeze proteins have the ability to bind to ice with high affinity and inhibit further crystal growth. The insect antifreeze protein from spruce budworm exhibits very high thermal hysteresis activity and is implicated in the protection of overwintering larvae from freezing. This protein has been crystallized in 20-25% polyethylene glycol (Mr 6000), 0.4 M NaCl, 0.1 M Tris-HCl, pH 8.5, by vapor diffusion using the hanging drop method. The resulting crystals are very thin (typically <0.01 mm in the shortest dimension), and only after repeated seeding could crystals be grown large enough for data collection using synchrotron radiation. The crystals belong to the monoclinic space group C2, with cell dimensions a = 82.28 A, b = 62.29 A, c = 63.63 A, and beta = 113.7 degrees. Molecules in the asymmetric unit are related by a twofold axis of symmetry with two molecules present. Native data to a resolution of 2.6 A have been collected with 90.3% completeness and a Rsym of 6.9%.     

Development of genome-wide insertion and deletion markers for maize,based on next-generation sequencing data

Background

Insertions and deletions (indels) are the most abundant form of structural variation in all genomes. Indels have been increasingly recognized as an important source of molecular markers due to high-density occurrence, cost-effectiveness, and ease of genotyping. Coupled with developments in bioinformatics, next-generation sequencing (NGS) platforms enable the discovery of millions of indel polymorphisms by comparing the whole genome sequences of individuals within a species.

Results

A total of 1,973,746 unique indels were identified in 345 maize genomes, with an overall density of 958.79 indels/Mbp, and an average allele number of 2.76, ranging from 2 to 107. There were 264,214 indels with polymorphism information content (PIC) values greater than or equal to 0.5, accounting for 13.39 % of overall indels. Of these highly polymorphic indels, we designed primer pairs for 83,481 and 29,403 indels with major allele differences (i.e. the size difference between the most and second most frequent alleles) greater than or equal to 3 and 8 bp, respectively, based on the differing resolution capabilities of gel electrophoresis. The accuracy of our indel markers was experimentally validated, and among 100 indel markers, average accuracy was approximately 90 %. In addition, we also validated the polymorphism of the indel markers. Of 100 highly polymorphic indel markers, all had polymorphisms with average PIC values of 0.54.

Conclusions

The maize genome is rich in indel polymorphisms. Intriguingly, the level of polymorphism in genic regions of the maize genome was higher than that in intergenic regions. The polymorphic indel markers developed from this study may enhance the efficiency of genetic research and marker-assisted breeding in maize.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1797-5) contains supplementary material, which is available to authorized users.     

An Antagonism Joint Action of Lead and Di-2-Ethylhexyl Phthalate Explains an Improved Ability of Learning and Memory after Combined Exposure in Weaning Rats

Lead and di-2-ethylhexyl phthalate (DEHP) are widely distributed in the environment, and their neurotoxicity has caused a widespread concern. The complexity of environmental exposure provides the possibility of their combined exposure. The present study aims to describe a joint neurotoxicity and clarify the potential mechanism after combined exposure to lead and DEHP. A 2 × 3 factorial design was used to analyze either single effects or their interaction by a subchronic lead and DEHP exposure model of the male weaning rats. Similar to the previous study, lead or DEHP single exposure showed an increased neurotoxicity. Interestingly, our neurobehavioral test showed the rats in the combined exposure groups had a better ability of learning and memory compared with the single-exposure ones. It seemed to reflect an antagonism joint action in neurotoxicity after combined exposure. The content of dehydroepiandrosterone (DHEA) in serum and the mRNA level of brain-derived neurotrophic factor (Bdnf) in the hippocampus showed a similar trend to the ability of learning and memory. However, there was insufficient evidence to support the joint action on some indexes of oxidative stress such as malondialdehyde (MDA), the ratio of reduced glutathione(GSH) to oxidized glutathione(GSSG), γglutamylcysteine synthetase (γ-GCS), glutathione-s transferase (GST), and nuclear factor E2-related factor 2 (Nrf2) mRNA expression in the hippocampus. In a word, our current study reminded a unique antagonism joint action of neurotoxicity after combined exposure to lead and DEHP, which may contribute to understanding some shallow mechanism of the joint toxicity due to the complexity of environmental pollutant exposure.

     

Metabolomic approach to optimizing and evaluating antibiotic treatment in the axenic culture of cyanobacterium Nostoc flagelliforme

The application of antibiotic treatment with assistance of metabolomic approach in axenic isolation of cyanobacterium Nostoc flagelliforme was investigated. Seven antibiotics were tested at 1–100 mg L^?1, and order of tolerance of N. flagelliforme cells was obtained as kanamycin > ampicillin, tetracycline > chloromycetin, gentamicin > spectinomycin > streptomycin. Four antibiotics were selected based on differences in antibiotic sensitivity of N. flagelliforme and associated bacteria, and their effects on N. flagelliforme cells including the changes of metabolic activity with antibiotics and the metabolic recovery after removal were assessed by a metabolomic approach based on gas chromatography–mass spectrometry combined with multivariate analysis. The results showed that antibiotic treatment had affected cell metabolism as antibiotics treated cells were metabolically distinct from control cells, but the metabolic activity would be recovered via eliminating antibiotics and the sequence of metabolic recovery time needed was spectinomycin, gentamicin > ampicillin > kanamycin. The procedures of antibiotic treatment have been accordingly optimized as a consecutive treatment starting with spectinomycin, then gentamicin, ampicillin and lastly kanamycin, and proved to be highly effective in eliminating the bacteria as examined by agar plating method and light microscope examination. Our work presented a strategy to obtain axenic culture of N. flagelliforme and provided a method for evaluating and optimizing cyanobacteria purification process through diagnosing target species cellular state.     

Network-assisted analysis to prioritize GWAS results: principles, methods and perspectives

Genome-wide association studies (GWAS) have rapidly become a powerful tool in genetic studies of complex diseases and traits. Traditionally, single marker-based tests have been used prevalently in GWAS and have uncovered tens of thousands of disease-associated SNPs. Network-assisted analysis (NAA) of GWAS data is an emerging area in which network-related approaches are developed and utilized to perform advanced analyses of GWAS data in order to study various human diseases or traits. Progress has been made in both methodology development and applications of NAA in GWAS data, and it has already been demonstrated that NAA results may enhance our interpretation and prioritization of candidate genes and markers. Inspired by the strong interest in and high demand for advanced GWAS data analysis, in this review article, we discuss the methodologies and strategies that have been reported for the NAA of GWAS data. Many NAA approaches search for subnetworks and assess the combined effects of multiple genes participating in the resultant subnetworks through a gene set analysis. With no restriction to pre-defined canonical pathways, NAA has the advantage of defining subnetworks with the guidance of the GWAS data under investigation. In addition, some NAA methods prioritize genes from GWAS data based on their interconnections in the reference network. Here, we summarize NAA applications to various diseases and discuss the available options and potential caveats related to their practical usage. Additionally, we provide perspectives regarding this rapidly growing research area.     

Regulation of alternative splicing of Bcl-x by IL-6, GM-CSF and TPA

The splicing of many alternative exons in the precursor messenger RNA (pre-mRNA) is regulated by extracellular factors but the underlying molecular bases remain unclear. Here we report the differential regulation of Bcl-x pre-mRNA splicing by extracellular factors and their distinct requirements for pre-mRNA elements. In K562 leukemia cells, treatment with interleukin-6 (IL-6) or granulocyte-macrophage colony stimulating factor (GM-CSF) reduced the proportion of the Bcl-xL variant mRNA while treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) had no effect. In U251 glioma cells, however, TPA efficiently increased the Bcl-xL level. These regulations were also seen for a transfected splicing reporter mini-gene. Further analyses of deletion mutants indicate that nucleotides 1-176 of the downstream intron are required for the IL-6 effect, whereas additional nucleotides 177-284 are essential for the GM-CSF effect. As for the TPA effect, only nucleotides 1-76 are required in the downstream intron. Thus, IL-6, GM-CSF and TPA differentially regulate Bcl-x splicing and require specific intronic pre-mRNA sequences for their respective effects.     

NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane subunit gp91^phox was dose-dependent. Meanwhile, the cytoplasmic subunit p47^phox was translocated to the cell membrane and localized with p22^phox and gp91^phox to form reactive NADPH oxidase. Our data suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.     

LC-MS/MS determination of helicid in human plasma and its application in pharmacokinetic studies

Helicid is a traditional Chinese medicine used to treat headache and insomnia with definite effects. To facilitate pharmacokinetic studies of helicid in man, a sensitive and specific LC-MS/MS method for the quantitative detection of helicid in human plasma was developed and validated. The method involved the addition of bergeninum as the internal standard (IS), protein precipitation, HPLC separation, and quantification by MS/MS system using negative electrospray ionization in the multiple reaction monitoring mode (MRM). The precursor→product ion transitions were monitored at m/z 282.8→120.9 for helicid and m/z 326.9→192.2 for the IS, respectively. The lower limit of quantification (LLOQ) was 0.2 μg/L. The calibration curves for helicid was linear over a concentration range of 0.2-20 μg/L. The intra- and inter-batch analyses of QC samples at 0.4, 2, 20 μg/L indicated good precision (%R.S.D. between 2.69 and 5.47%) and accuracy (between 96.15 and 105.05%). The helicid was stable in human plasma stored at room temperature for at least 24h, 4°C for at least 24h, -20°C for at least 1 month, and for routine three freeze-thaw cycles. This accurate and specific assay provides a useful method for evaluating the pharmacokinetic profile of helicid in humans.